Modelling altered signalling of G-protein coupled receptors in inflamed environment to advance drug design

We previously reported the successful design, synthesis and testing of the prototype opioid painkiller NFEPP that does not elicit adverse side effects. The design process of NFEPP was based on mathematical modelling of extracellular interactions between G-protein coupled receptors (GPCRs) and ligands, recognizing that GPCRs function differently under pathological versus healthy conditions. We now present an additional and novel stochastic model of GPCR function that includes intracellular dissociation of G-protein subunits and modulation of plasma membrane calcium channels and their dependence on parameters of inflamed and healthy tissue (pH, radicals). The model is validated against in vitro experimental data for the ligands NFEPP and fentanyl at different pH values and radical concentrations. We observe markedly reduced binding affinity and calcium channel inhibition for NFEPP at normal pH compared to lower pH, in contrast to the effect of fentanyl. For increasing radical concentrations, we find enhanced constitutive G-protein activation but reduced ligand binding affinity. Assessing the different effects, the results suggest that, compared to radicals, low pH is a more important determinant of overall GPCR function in an inflamed environment. Future drug design efforts should take this into account.

transfected with 1 µg per 200 µl transfection mix of each plasmid containing the different cDNAs using X-tremeGENE HP DNA Transfection Reagent (Roche, Mannheim, Germany) following the manufacturer's instructions. For stable transfection, pcDNA TM 3.1+ carrying the rat MOR provided by Christian Zöllner (University Hamburg, Germany) was linearised with restriction enzyme Bg1II (NEB, Frankfurt, Germany), and linearisation was verified by agarose gel electrophoresis. After 48 h, the medium containing the transfection reagent was removed and replaced by complete DMEM with 10% fetal bovine serum and penicillin/streptomycin (100 U/ml). Successfully transfected cells were selected by adding G418 (500 µg/ml) into medium that was renewed every 2 to 3 days. Monoclonal cell lines were then created 17 days post transfection by picking single colonies of stably transfected cells using a 100 µl pipette and transferring them to poly-L-Lysine coated wells of a 96-well plate. Cells were grown to confluence and successively transferred to larger culture flasks in the continued presence of 500 µg/ml G418. Antibiotic concentration was reduced to 100 µg/ml when the cells were moved to 75 cm 2 culture flasks. Monoclonal cell lines were further characterised based on immunocytochemistry, MOR mRNA expression, subjective impression of cell growth and overall cell morphology, as described previously 3 . Stably transfected cell lines were cultured for a maximum of 23 passages.
Protein concentrations were determined with the Bradford assay using Coomassie Brilliant Blue G-250 dye (Bio-Rad Laboratories GmbH, München, Germany) that shifts absorption from 465 to 595 nm upon binding to proteins. The relationship between measured absorbance and protein concentration was established based on a standard curve obtained from fixed protein solutions of known composition and concentration. These measurements were performed in duplicates using Bio-Rad Protein Assay Dye Reagent Concentrate with Bio-Rad Protein Assay Standard II (Bio-Rad). Samples with unknown concentrations, standards and dye reagent concentrate were diluted according to the manufacturer's instructions, thoroughly mixed, and incubated for 5 min at room temperature. Absorption at 595 nm was measured in triplicates with a spectrophotometer. Generation of linear standard curves and interpolation of total protein concentration was performed by the device's inbuilt software. A standard curve was generated for every experiment.
Membrane fractions were prepared from transfected HEK293 cells as described previously 44 . The cells were grown in 175 cm 2 tissue culture flasks to approximately 90% confluence. Cells were then washed with Tris buffer (50 mM, Trizma preset crystals, pH 7.4; Sigma Aldrich), harvested with a scraper, homogenised using a mechanical disperser (Dispergierstation T8.10, IKA-Werke, Staufen, Germany) at maximum speed for 10 s and centrifuged at 42K×g for 20 min at 4°C (Avanti JXN-26 ultracentrifuge, Beckmann Coulter, Krefeld, Germany). Cellular pellets including membranes with embedded and anchored proteins were then resuspendend in Tris buffer for washing to separate them from cytosolic components by homogenisation and centrifugation at the same settings. Supernatants were discarded and the pellets were stored at -80°C. On the day of usage, the pellets were thawed on ice in Tris buffer and homogenised. Total protein concentrations were determined as described above and homogenates were split according to the number of conditions tested in respective assay buffers.
The The data on dissociation of G-protein subunits, as measured by FRET, were extracted from 3 . Methodological details are described in 3 .

Data Analysis
Experimental designs were randomised to compensate for the position effects on plates or filter apparatus and unequal sample processing time. Sample sizes were calculated using the G * Power 3.1.2 program with α < 0.05, a power of 80% and a defined effect size (derived from pilot experiments). Analysis of concentration-response relationship was performed with simple linear regression using the GraphPad Prism 9 program (GraphPad, San Diego, USA) where y = [ 35 S]-GTPγS bound and x = 2/4